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Chronic hepatitis B virus (HBV) infection can cause cirrhosis and hepatocellular carcinoma and is therefore a serious public health problem. Infected patients are currently treated with nucleoside/nucleotide analogs and interferon α, but this approach is not curative. Here, we screen 978 FDA-approved compounds for their ability to inhibit HBV replication in HBV-expressing HepG2.2.15 cells. We find that ciclopirox, a synthetic antifungal agent, strongly inhibits HBV replication in cells and in mice by blocking HBV capsid assembly. The crystal structure of the HBV core protein and ciclopirox complex reveals a unique binding mode at dimer-dimer interfaces. Ciclopirox synergizes with nucleoside/nucleotide analogs to prevent HBV replication in cells and in a humanized liver mouse model. Therefore, orally-administered ciclopirox may provide a novel opportunity to combat chronic HBV infection by blocking HBV capsid assembly. Current treatments for chronic hepatitis B virus (HBV) infection are not curative. Here, the authors show that an antifungal drug, ciclopirox, inhibits HBV capsid assembly and synergizes with nucleoside/nucleotide analogs to prevent HBV replication in cells and in a humanized liver mouse model.

N6A-methyladenosine (m6A) post-transcriptional modification, the most abundant internal RNA modification, is catalyzed by the METTL3-14 methyltransferase complex. Recently, attention has been drawn to the METTL3-14 complex regarding its significant roles in the pathogenesis of acute myeloid leukemia (AML), attracting the potential of novel therapeutic targets for the disease. Herein, we report the identification and characterization of eltrombopag as a selective allosteric inhibitor of the METTL3-14 complex. Eltrombopag exhibited selective inhibitory activity in the most active catalytic form of the METTL3-14 complex by direct binding, and the mechanism of inhibition was confirmed as a noncompetitive inhibition by interacting at a putative allosteric binding site in METTL3, which was predicted by cavity search and molecular docking studies. At a cellular level, eltrombopag displayed anti-proliferative effects in the relevant AML cell line, MOLM-13, in correlation with a reduction in m6A levels. Molecular mechanism studies of eltrombopag using m6A-seq analysis provided further evidence of its cellular function by determining the hypomethylation of leukemogenic genes in eltrombopag-treated MOLM-13 cells and the overlapping of the pattern with those of METTL3-knockdown MOLM-13 cells. In conclusion, eltrombopag was first disclosed as a functional METTL3-14 allosteric inhibitor in AML cells, which could be utilized for the further development of novel anti-AML therapy.

(1) Background: The microRNA (miR)-directed control of gene expression is correlated with numerous physiological processes as well as the pathological features of tumors. The focus of this study is on the role of miRs in the regulation of RSU1 and proteins in the IPP (integrin linked kinase, PINCH and parvin) complex. Because the IPP adaptor proteins link β integrins to actin cytoskeleton, and the RSU1 signaling protein connects the complex to the activation of cJun, ATF2 and the transcription of PTEN, their reduction by miRs has the potential to alter both adhesion and survival signaling. (2) Methods: Multiple database analyses were used to identify miRs that target RSU1 and PINCH1. miR transfection validated the effects of miRs on RSU1, PINCH1 and downstream targets in breast cancer cell lines. (3) Results: The miRs targeting RSU1 mRNA include miR-182-5p, -409-3p, -130a-3p, -221-3p, -744-5p and -106b-5p. Data show that miR-182-5p and -409-3p reduce RSU1, PINCH1 and inhibit the ATF2 activation of PTEN expression. miR-221-3p and miR-130a-3p target RSU1 and PINCH1 and, conversely, RSU1 depletion increases miR-221-3p and miR-130a-3p. (4) Conclusions: miRs targeting RSU1 and PINCH1 in mammary epithelial or luminal breast cancer cell lines reduced RSU1 signaling to p38 MAP kinase and ATF2, inhibiting the expression of PTEN. miR-221-3p, known to target PTEN and cell cycle regulators, also targets RSU1 and PINCH1 in luminal breast cancer cell lines.

Surgical excision (Mohs micrographic surgery) is the standard procedure to treat a melanoma, in which an in situ histologic examination of sectioned skin is carried out repeatedly until no cancer cells are detected. The possibility to identify melanoma from the surrounding skin by femtosecond laser-induced breakdown spectroscopy (fs-LIBS) is investigated. For experiments, melanoma induced on a hairless mouse by injection of B16/F10 murine melanoma cell was sampled in the form of frozen tissue sections as in Mohs surgery and analyzed by fs-LIBS (λ  =  1030  nm, τ  =  550  fs). For analysis, the magnesium signal normalized by carbon intensity was utilized to construct an intensity map around the cancer, including both melanoma and surrounding dermis. The intensity map showed a close match to the optically observed morphological and histological features near the cancer region. The results showed that when incorporated into the existing micrographic surgery procedure, fs-LIBS could be a useful tool for histopathologic interpretation of skin cancer possibly with significant reduction of histologic examination time.

Tamoxifen (TAM) is the standard anti-hormonal therapy for estrogen receptor-positive breast cancer. However, long-term TAM therapy can make acquisition of TAM resistance and there are still no solutions to treat TAM-resistant breast cancer. In this study, we found that protein and mRNA expression of the P2X purinoreceptor 7 (P2X7) was higher in tamoxifen resistant MCF-7 (TAMR-MCF-7) cells than in control MCF-7 cells. P2X7 inhibition potently inhibited the migration of TAMR-MCF-7 cells and the liver metastasis burden of TAMR-MCF-7 cells in the spleen-liver metastasis experiment. However, the P2X7 antagonist did not affect protein expression of matrix metalloproteinase (MMP)-2, MMP-9, and epithelial-mesenchymal transition markers. Here our data indicate a link between small extracellular vesicles (sEV) and P2X7, and suggest a new mechanism of metastasis in TAM-resistant breast cancer cells through P2X7 receptors. The migration of TAMR-MCF-7 cells was increased in a concentration-dependent manner by purified sEV treatment. The number of secreted sEVs and the protein levels of CD63 in TAMR-MCF-7 cells were decreased by the P2X7 antagonist, showing that P2X7 influences the production of sEV. Our results suggest that inhibiting the P2X7 could be considered for metastasis prevention in TAM-resistant cancer patients.

P2X3 receptors (P2X3R) are ATP-gated ion channels predominantly expressed in C- and Aδ-fiber primary afferent neurons and have been introduced as a novel therapeutic target for neurological disorders, including neuropathic pain and chronic cough. Because of its localized distribution, antagonism of P2X3R has been thoroughly considered, and the avoidance of issues related to CNS side effects has been proven in clinical trials. In this article, benzimidazole-4,7-dione-based derivatives were introduced as a new chemical entity for the development of P2X3R antagonists. Starting from the discovery of a hit compound from the screening of 8364 random library compounds in the Korea Chemical Bank, which had an IC50 value of 1030 nM, studies of structure–activity and structure–property relationships enabled further optimization toward improving the antagonistic activities as well as the drug’s physicochemical properties, including metabolic stability. As for the results, the final optimized compound 14h was developed with an IC50 value of 375 nM at P2X3R with more than 23-fold selectivity versus P2X2/3R, along with properties of metabolic stability and improved solubility. In neuropathic pain animal models evoked by either nerve ligation or chemotherapeutics in male Sprague-Dawley rats, compound 14h showed anti-nociceptive effects through an increase in the mechanical withdrawal threshold as measured by von Frey filament following intravenous administration.

Glioblastoma (GBM) poses a serious challenge due to its aggressive nature and poor prognosis. Tumor mesenchymal stem-like cells (tMSLCs) secrete complement component 5a (C5a), altering the tumor microenvironment (TME) and promoting tumor progression. This study investigated W54011, a C5a antagonist, to counteract C5a-induced malignancy in GBM tumorspheres. We assessed GBM tissues for C5a receptor 1 (C5aR1) expression using gene profiling and survival analysis. GBM tumorspheres were cultured in C5a-enriched conditioned medium (CM) from tMSLCs to induce tumor stimulation. We evaluated proliferation, invasion, and stemness of GBM tumorspheres using WST/ATP, matrigel invasion assay, and limiting dilution assays. Results were validated via western blotting and RNA sequencing. Additionally, findings were corroborated in an in vivo xenograft mouse model. High C5aR1 expression correlated with increased TME, inflammation-related gene expression, and poorer patient outcomes. CM treatment increased GBM tumorsphere proliferation, invasion, and stemness, which were reversed by W54011. CM also induced the epithelial-mesenchymal transition, whereas W54011 restored spherical morphology and induced apoptosis. In xenograft models, CM-treated GBM tumorspheres led to larger tumors and decreased survival, whereas W54011 decreased tumor size and improved survival. This study suggests a potential role for C5a in GBM progression and supports further investigation of W54011 as a therapeutic candidate.

The P2X7 receptor is an ATP-gated ion channel that activates inflammatory pathways involved in diseases such as cancer, atherosclerosis, and neurodegeneration. However, despite the potential benefits of blocking overactive signaling, no P2X7 receptor antagonists have been approved for clinical use. Understanding species-specific pharmacological effects of existing antagonists has been challenging, in part due to the dearth of molecular information on receptor orthologs. Here, to identify distinct molecular features in the human receptor, we determine high-resolution cryo-EM structures of the full-length wild-type human P2X7 receptor in apo closed and ATP-bound open state conformations and draw comparisons with structures of other orthologs. We also report a cryo-EM structure of the human receptor in complex with an adamantane-based inhibitor, which we leverage, in conjunction with functional data and molecular dynamics simulations, to design a potent and selective antagonist with a unique polycyclic scaffold. Functional and structural analysis reveal how this optimized ligand, termed UB-MBX-46, interacts with the classical allosteric pocket of the human P2X7 receptor with subnanomolar potency and high selectivity, revealing its significant therapeutic potential. Comparison between structures of human, mouse and rat P2X7 receptors define ortholog-specific pharmacology and facilitated structure-based drug design of UB-MBX-46, an antagonist that selectively inhibits the human P2X7 receptor with sub-nanomolar potency.

BackgroundWe aimed to investigate the prevalence of dysautonomia in complex regional pain syndrome (CRPS) via the combined autonomic nervous system (ANS) function tests, including the deep breathing test (DBT), orthostatic test (OST) and sympathetic skin response (SSR).MethodWe retrospectively examined 263 patients who underwent the combined ANS tests to evaluate CRPS between August 2013 and December 2016. Based on the Budapest clinical criteria, patients were stratified into confirmed-CRPS or suspected-CRPS groups. We performed binary logistic regression analysis using the inverse probability of treatment weighting to investigate the association between the tests and CRPS. Sensitivity and specificity were calculated to assess the diagnostic performance of the ANS tests for CRPS. We compared the results of these tests between the outcomes of sympathetic nerve blocks (SNBs).ResultsAmong 247 patients, finally included in this study, 199 patients (80.6%) were diagnosed with CRPS. Abnormal results of overall or each ANS function test showed significant associations with CRPS, excluding OST (overall abnormality: OR 2.44, 95% CI 1.51 to 3.95; p<0.001; DBT: OR 2.57, 95% CI 1.23 to 5.38, p=0.013; OST: OR 1.88, 95% CI 0.92 to 3.84, p=0.085; SSR: OR 2.71, 95% CI 1.38 to 5.32, p=0.004). However, their prevalence in CRPS and their sensitivities for CRPS were low (overall abnormality: 26.1%; each test: <15%). No significant association existed between dysautonomia and SNB outcomes.ConclusionDysautonomia, as evaluated using the combined ANS tests, were observed in a small portion of patients with CRPS. The diagnostic performances of these tests for CRPS were inadequate for clinical purposes.

Cladding for dome roofs is often made of membrane materials that are light and easy to install. Due to these characteristics, wind damage to dome roof cladding is very common. In particular, open or retractable dome roofs are prone to wind damage because of inadequacies in wind load calculations. In this study, the wind pressure characteristics of a dome with a central opening were investigated. Wind tunnel tests were performed, and the pressure distribution was investigated by analyzing external and internal pressure coefficients. Based on the experimental results, the peak net pressure coefficients for the cladding design of a dome roof with a central opening were proposed. For the external peak pressure coefficients, the values of leeward regions were similar despite height–span ratios and turbulence intensity values. For the internal peak pressure coefficients, negative pressure was dominant, and the coefficients were not significantly affected by changes in height–span ratio. This tendency locally increased the negative peak net pressure, in which the load acts in the upward direction, and relatively significantly increased the positive peak net pressure, in which the load acts in the downward direction.